Dear Researchers,
I am regularly preparing 1/2 MS liquid media for transformation protocol. It appears good before autoclaving but often gets precipitated (white in color) after autoclaving. Is it ok to use the media for experiments or will it create problem? Can anyone tell the reason and solution for this problem? Thanks in advance.
Just control your solidifier (agar, gelrite etc.) and be sure that your bottles having liquid media are clean or not.
Hello Prem Kumar G ,
Precipitation in tissue culture media usually observed with time. However, most of the time it was observed that ready-made old media, addition of higher concentration of salts in stock solution than its solubility, preparation of MS major minor stock solution with distilled water rather than double distilled water and measurement and preparation error (like preparation of medium with undissolved or poorly dissolved salts of CaCl2, magnesium and pH adjustment) significantly contributes to the precipitation while preparation. pale yellow precipitation in media formed due to inevitable oxidation of ferrous ions to ferric ions. Although, no negative effect reported by any researcher to the best of my knowledge. I suggest you to go with all parameters and review them, hope you soon get rid of it.
wishing you good luck
prem,
As of the data provided by you, Some times it is common to observe the precipitation of MS liquid medium (1/2 or full strength). Even during our tissue culture experimental studies we observed same problem.
The precipitation might be due
a. distilled water,
b. Use of old MS media (ready one)
c. The pH & very high temperature during autoclave.
d. improper homogenization.
e. As suggested by Mustafa, the presence of residual salts or detergents in the glassware's also contributes.
If you are preparing through stock solution & preparing, autoclave FeNa-EDTA and KH2PO4 separately and add (Schenk et al., 1991).
As of various studies it can be used for the experiments. If precipitation is more its better to use freshly prepared one with all caution.
Sigma data sheet: Analysis of precipitates indicated a predominance of iron, phosphate, and zinc. The probable cause of the precipitates is the inevitable oxidation of ferrous ions to ferric ions and the presence of unchelated ferric ions. When the solubility of ferric phosphate is exceeded precipitation occurs. There are no reports of detrimental effects on growth and development in plant tissue culture due to the precipitates.
http://www.sigmaaldrich.com/technical-documents/protocols/biology/media-preparation.html
https://www.ncbi.nlm.nih.gov/pubmed/24221488
Much more thanks to all. Now I got idea about the cause for the problem.
Precipitation in the media usually occurs due to the poorly dissolved salts of calcium chloride, occurs in both manual as well as ready made (old) MS media. It occurs not only in autoclaved media but also in case of un-autoclaved media. In case of manually prepared liquid media, try to dissolve calcium salts completely or filtering micro salt solutions before using the stock for preparation of liquid media will be beneficial. Precipitates irrespective of their color are misinterpreted as bacterial contamination even though they do not posses any negative effect on callus growth.
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